Bioactive peptide complexes

ABSTRACT

Herein disclosed are bioactive peptides and proteins having immunomodulating and antiviral activity, more particularly histidine-rich bioactive peptide complexes having the following structural formula (SEQ ID NO: 29): 
                         
wherein: X 1  is preferably absent or is selected from among Gln, Ser, Asn, Val, Ala, Phe, and Asp, and R 1  and R 2  are peptide chains optionally contain the amino acid residues His and/or Cys that interact with transition metal ions. Whereas R 1  is preferably selected from among His-Gly-Val-Ser-Gly(SEQ ID NO: 30), CysVal-Val-Thr-Gly- (SEQ ID NO: 31), Cys-Gly-, Val-Ser-Gly-, and His-Gly- or alternatively is absent, R 2  is preferably selected from among -Val-His-Gly, -Val-Phe-Val, -Val-His, -Val-Asp or alternatively is absent. Such histidine-rich peptide complexes, primarily alloferon family peptides such as Alloferon-1 (SEQ ID NO: 1) with Zn ions, enable the creation of drugs with a targeted mechanism of action, and the design thereof with regard to understanding of drug target structure.

PRIORITY

This application is a divisional of U.S. application Ser. No. 14/401,353 filed Nov. 14, 2014, which, in turn, corresponds to the U.S. National Phase of International Application No. PCT/RU2012/000405 filed May 21, 2012, the entire contents of which are hereby incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 12, 2015, is named LNK_157US_SL.txt and is 10,981 bytes in size.

FIELD OF THE PRESENT INVENTION

The present invention refers to proteins and bioactive peptides with immunomodulating and antiviral activity.

BACKGROUND OF THE PRESENT INVENTION

Peptide, polypeptide and protein-based compounds used in medicine as antiviral drugs are known. Among type I interferon inducers (IFI) they are known as high-molecular compounds [F. I. Yershov, O. I. Koselev. Interferons and their inducers from the molecule to the drug—, M.: Publ. House. Geotar-Media, 2005-P. 356], [Berg K., Bolt G., Andersen H., Owen T C. Zink potentiates the antiviral action of human IFN-alpha tenfold. J. Interferon Cytokine Res, 2001, July; 21(7):471-4], as low-molecular inducers. From the latter, first of all, native drug cycloferon and American drug imiquimod should be noted. These drugs refer to acridone and benzimidazole derivatives, respectively. For imiquimod and close derivatives, Toll-like type of receptors is known, with which this group of drugs interacts causing IFN-α synthesis induction in various cells [F. I. Yershov., O. I. Kiselev. Interferons and their inducers (from the molecule to the drug) M.: Publ. House. Geotar-Media, 2005.-P. 356].

Bioactivity of low-molecular peptides is widely known. First of all, this refers to animal and plant origin peptides with antibacterial activity [Boman H. Peptide antibiotics and their role in innate immunity. Anu. Rev. of Immunol., 1995, Vol. 13, p. 61-92]. However, a number of peptides possessing direct antiviral and antitumour action has been described [Akiyama N., Hijikata M., Kobayashi A., Yamori T., Tsuruo T., Natori S. Anti-tumor effect of N-β-alanyl-5-S-glutathionyl dihydroxyphenylalanine (5-S-GAD) a novel anti-bacterial substance from an insect. Anticancer Research, 2000, Vol. 20, p. 357-362].

Peptides of amphibians and insects take a special place here [Bulet P., Hetru C., Diamarcq J., Hoffmann D. Antimicrobial peptides in insects: structure and function. Devel. Comp. Immunol., 1999, Vol. 23, p. 329-344, Chinchar V. G., Wang J., Murti G., Carey C., Rolling-Smith L. Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin. Virology, 2001, Vol. 288, p. 351-357].

Immunomodulating peptides—alloferons are known (patent of the RF U.S. Pat. No. 2,172,322). Treatment of viral infections is the main area of application for alloferons. Alloferons are the closest analogues of the present invention regarding chemical structure and mode of action.

It should be noted, that inventors of the patent U.S. Pat. No. 2,172,322 only consider variations of primary alloferon structure and do not place key value to histidine residues distribution.

Moreover, alloferons should be referred to quite “weak” interferon inducers, which is evident when comparing their activity with cycloferon.

At the same time, alloferons structure stands out with regular histidine residues arrangement and frequent glycine residues. Enhancement of alloferons structure is possible towards giving them tertiary structure elements, for instance, by introduction of metal ions.

Hemin-peptide and its pharmaceutically acceptable salts with virucidal and antiviral action, containing metal ions, where Zn, Cu, Fe, Mn can be used, is also known. (Patent of the RF U.S. Pat. No. 2,296,131). However, this compound refers to the second class of peptides and is not an immune modulator.

Peptide complexes with Zn⁺⁺ ion, with elements of organized tertiary structure and activity of first type interferon inducers, are not described in the literature.

Need for modification of histidine-containing peptides with Zn⁺⁺ ion is driven by the following causes:

1. Bioactive short peptides have disorganized type of secondary structure inevitably reducing their bioactivity, interactability with other macromolecules, metabolic stability.

2. Biological and pharmacological activity of peptides largely depends on transport efficiency to cells. Making peptide structure compact increases effectiveness of their translocation through membranes and, subsequently, pharmacological activity [Leng Q., Mixson J. Modified branched peptides with histidine-rich tail enhance in vitro gene transfection. Nucl. Acids. Res., 2005, Vol. 33, e40].

3. Formation of histidine-containing peptide complexes with Zn⁺⁺ ion results in fundamental changes of peptides properties, making them identical with domains of transcriptional activators of viruses and cells.

SUMMARY OF THE PRESENT INVENTION

The objective of the present invention is to develop peptide complexes organized in three-dimensional structure. The designed complexes possess high binding ability with other molecular groups and display wide spectrum of pharmacological action, including type I IFN induction and act on various levels of cellular functions, allowing to create new drugs for prevention and treatment of viral infections based on them.

The new family of bioactive peptides has been developed based on the known peptides, enriched with histidine residues, alloferons and their homologues using Zn-finger of protein domains with known functions as a prototype. Alloferons are used as a peptide matrix 6 to 35 amino acid residues long. In this way engineered peptides are able to form complexes with Zn⁺⁺ ion, creating oligomers and aggregates, and regarding structural and biological properties they meet the requirements of immune modulators.

Present peptide complexes have three-dimensional structure and are described by the following structural formula (SEQ ID NO: 23):

where: X₁ is absent or contains not less than 1 amino acid; and R1 and R2 are peptide chains containing amino acid residues that interact with transition metal ions, with R1 containing up to 5 amino acid residues or absent; R2 contains up to 3 amino acid residues or absent.

Ability of natural peptides, enriched with histidine residues, to bind with metal ions has been proved in a number of studies [Hua Zhao H., and Waite J. H. Proteins in Load-Bearing Junctions: The Histidine-Rich Metal-Binding Protein of Mussel Byssus, Biochemistry. 2006, 45(47): 14223-14231].

BRIEF DESCRIPTION OF THE FIGURES

Essence of invention is explained with the data from the schemes and figures:

FIG. 1. Consensus sequence analysis of alloferon family peptides. FIG. 1 discloses SEQ ID NOS 1-21, 25, 1-21, 26, 1-21, and 25, respectively, in order of appearance.

FIGS. 2A and 2B. A1 polypeptide computer model. FIG. 2A discloses SEQ ID NO: 1.

FIGS. 3A and 3B. Theoretical options of structures of A1 complexes with Zn⁺⁺ ion. FIG. 3A discloses SEQ ID NO: 1 and FIG. 3B discloses SEQ ID NOS 28 and 28, respectively, in order of appearance.

FIG. 4. Binding kinetics of alloferon A1 with Zn⁺⁺ by light-scattering method.

FIG. 5. Peptide A1 binding analysis with Ni⁺⁺ balanced HiTrap adsorbent.

FIG. 6. Type I interferons induction.

FIG. 7. Protective effect of the studied drugs in case of lethal grippal infection in mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Alloferon 1 (SEQ ID NO 1) peptide, presented in the Table 2, has been used as a base structure during development of the present invention. Alloferon 1 was synthesized by solid-phase synthesis method and used to study bioactivity of the present peptides. The studies, the findings of which were presented in examples below, demonstrated that this peptide has ability to form complexes with transition metals, is interferon inducer and possesses antiviral activity.

Databases computer analysis of the proteins and peptides structure and properties found that this compound refers to the novel family of bioactive peptides. Histidine and glycine-rich polypeptides with introduced metal ions possess immune modulating and antiviral activity with zinc ions potentiating their bioactivity.

Synthesis of the present sequence of peptides has been performed in solid-phase peptides synthesis using Boc/Bzl strategies of phenyl acetamide methyl polymer (PAM). Peptides were isolated on Coupler-250 and Applied Biosystems 430A peptide synthesizers.

Tert-Butoxycarbonilamino group was used for temporary protection of α-amino groups removed with trifluoroacetic acid. Benzyl and acyl types safety groups have been used for suppression of lateral radicals of trifunctional amino acids: dinitrophenyl for histidine, mesitylenesulfonyl for arginine, 2-chlorbenzyloxycarbonyl for lysine, fromyl for tryptophan, 2,6-dychlorbenzyl for tyrosine, O-benzyl ethers for threonine and serine. Methionine was administered in condensation in the form of sulphoxy derivative.

Removal of temporary protection groups was performed with undiluted trifluoroacetic acid, and neutralization—by in situ method, adding N,N′-diisopropylethylamine at condensation stage directly into reaction mixture.

The program for addition of one amino acid residue during peptidyl-polymer chain elongation in an amount of total content of acylamino acid on the 0.2 mmol polymer is given in the table. Preactivation of carboxy component was performed within 30 minutes using hydroxybenzotriazole and diisopropylcarbodiimide. Under such conditions of synthesis in all the cases after addition of needed volume of amino acid residues, relevant to peptide fragment sequence, satisfactory peptidyl-polymer increment was reached.

Removal of side protection groups and peptide elimination from resin was performed under the action of anhydrous hydrogen fluoride in the presence of scavengers, mainly, m-cresol. During such treatment, all the side protection groups were removed and peptide was eliminated from high-molecular matrix, release time fluctuated from one to one and a half hour.

To prevent from adverse reactions during methionine-containing peptides synthesis, (in particular, sulphur alkylation with tert-butyl radical, and its partial oxidation during peptide chain elongation) methionine residues are smoothly added into peptidylpolymer sequence in the form of sulphoxy derivative, which at the end stages of peptide release was recovered to methionine. This recovery reaction had satisfactory results when treated with ammonium iodide or with completely released peptide, or at the stage, when peptide was still at the resin.

TABLE 1 Program for addition of one amino acid residue Reagents Repetition volume, No. Operation Reagents factor Time, min mL 1. Removal of Trifluoroacetic acid 1 2 5 Rec-protection (release) 2. Metronomic Trifluoroacetic acid 1 2 5 release 3. Washing Dymethylformamide 3 1 10 4 Condensation 1.0 mmol of oxybenzotrisol 1 20 5 ether of the relevant amino acid derivative + Diisopropylethylamine (0.7 mmol in dymethylformamide) 5. Washing Dymethylformamide 3 1 10 6. Washing Methylene dichloride 3 1 10 7. Ninhydrin test* *condensation was repeated in case of positive ninhydrin test

All synthesized peptide drugs were purified using preparative reverse-phase liquid chromatography at the column Dynamax 60 A, 22.5×250 mm (liquid chromatograph Gilson, France) and are characterized by findings of hydrolysate peptides amino acid analysis after hydrolysis with methanesulfonic acid in the presence of tryptamine (amino acid analyzer Alpha Plus, LKB, Sweden).

EXAMPLES

The following examples prove the possibility to accomplish the object of invention.

Example 1. Analysis of Structure and Consensus Sequences of Alloferon Family Peptides

BioEdit v.7.09 Ibis Biosciences (US) software was used for consensus sequence analysis of alloferon peptides families. Alloferon amino acid sequences homology is presented in the table 2.

TABLE 2 Alloferon sequence homology peptide 1 2 3 4 5 6 7 8 9 10 11 12 13 14 SEQ ID NO 1 His Gly Val Ser Gly His Gly Gln His Gly Val His Gly Alloferon 1 SEQ ID NO 2 Cys Val Val Thr Gly His Gly Ser His Gly Val Phe Val Alloferon 10 SEQ ID NO 3 Ile Ser Gly His Gly Gln His Gly Val Pro Alloferon 11 SEQ ID NO 4 Cys Gly His Gly Asn His Gly Val His Alloferon 12 SEQ ID NO 5 Ile Val Ala Arg Ile His Gly Gln Asn His Gly Leu Alloferon 13 SEQ ID NO 6 His Gly Ser Asp Gly His Gly Val Gln His Gly Alloferon 14 SEQ ID NO 7 Phe Gly His Gly His Gly Val Alloferon 15 SEQ ID NO8 His Gly Asn His Gly Val Leu Ala Alloferon 16 SEQ ID NO 9 His Gly Asp Ser Gly His Gly Gln His Gly Val Asp Alloferon 17 SEQ ID NO 10 His Gly His Gly Val Pro Leu Alloferon 18 SEQ ID NO 11 Ser Gly His Gly Ala Val His Gly Val Met Alloferon 19 SEQ ID NO 12 Gly Val Ser Gly His Gly Gln His Gly Val His Gly Alloferon 2 SEQ ID NO 13 Tyr Ala Met Ser Gly His Gly His Gly Val Phe Ile Alloferon 20 SEQ ID NO 14 Val Ser Gly His Gly Gln His Gly Val His Alloferon 3 SEQ ID NO 15 Ser Gly His Gly Gln His Gly Val Alloferon 4 SEQ ID NO 16 Pro Ser Leu Thr Gly His Gly Phe His Gly Val Tyr Asp Alloferon 5 SEQ ID NO 17 Phe Ile Val Ser Ala His Gly Asp His Gly Val Alloferon 6 SEQ ID NO 18 Thr His Gly Gln His Gly Val Alloferon 7 SEQ ID NO 19 His Gly His Gly Val His Gly Alloferon 8 SEQ ID NO 20 Leu Ala Ser Leu His Gly Gln His Gly Val Alloferon 9 SEQ ID NO 21 His Gly Tyr Thr Ser His Gly Ala His Gly Val Gemagglutin 377-388 SEQ ID NO: 24 His Gly His Gly Consensus sequence SEQ ID NO: 24 R1 His Gly X1 His Gly R2 Structural formula

Patent of the RF U.S. Pat. No. 2,172,322 illustrates alloferons sequence without consensus sequence presentation, which makes it impossible to precisely estimate core-heart part of peptides and separate significant modifications from insignificant.

Resulting from the analysis, alloferon family can be divided into 3 families with consensus sequences:

SGHGQ-HGV (SEQ ID NO: 25), VSGHGQ-HGV (SEQ ID NO: 26), SGHGQ-HGV (SEQ ID NO: 25), which is substantiated with the given computer estimations (FIG. 1) of alloferon families peptides sequences.

Example 2. Peptide A1 Computer Modeling (Tertiary Structure Analysis)

To understand short peptides structure, it is possible to use computer modeling, allowing to estimate peptide structure in whole and its separate domains. In particular, we needed to estimate potential for creation of the present peptides complexes with Zn⁺⁺ ion. For this, computer modeling of A1 peptide with the following structure was performed: His-Val-Ser-His-Gly-Gln-His-Gly-Val-His-Gly (A1) (SEQ ID NO: 27). Simple A1 complex buildup with Zn⁺⁺ ion allows to demonstrate peptide loop formation, stabilized with coordinate bonds of histidine residues with Zn⁺⁺ ion.

A1 peptide computer modeling (FIGS. 2A and 2B) showed that short peptide forms relax loop, where Zn⁺⁺ ion can interact with histidine residues accessible for interaction. In this case, general polypeptide structure fits the possibility to form Zn⁺⁺ ion complex at least with three histidine residues in loci 1.6 and 9.

The simplified model (FIGS. 3A and 3B) Zn-A1 shows that significant portion of glycine residues is located in the N-end part of molecule. This corresponds to secondary structure of beta layers type. C-end part has alpha-helical structure with inside-exposed imidazole rings of histidine accessible for interaction with Zn⁺⁺ ion.

The Figure illustrates example with Zn⁺⁺. Zn⁺⁺ can be located virtually in any position.

A—intramolecular complex Zn-A1, organized as a loop.

A—intermolecular complex Zn-A1, organized as a dimer. Aggregation can be performed by adding new A1 molecules due to intermolecular fusion of Zn⁺⁺ ion in <<a>> and <<b>> regions or in the center of linear polypeptide with interaction of Zn⁺⁺ and histidine residues in positions 6 and 9.

When analyzing A1 structure, high content and regular arrangement of histidine residues drives attention. FIGS. 2A and 2B show that A1 polypeptide forms almost perfect saddle-like structure. Histidine residues 1, 6 and 9 are most accessible for interaction with Zn⁺⁺ ion in this confirmation.

In this case significant conclusion can be made that complex formations with peptide excess comparing to Zn⁺⁺ can result in formation of intermolecular aggregates (FIG. 2B) Such structural transition fundamentally changes peptides properties making their structure, needed for bioactivity, compact, which was demonstrated in numerous studies [Rydengard V., Nordahl E. A., Schmidtchen A. Zinc potentiates the antibacterial effects of histidine-rich peptides against Enterococcus faecalis. FEBS Lett., 2006, Vol. 273, p. 2399-2406].

Example 3. Alloferon and its Closest Analogues are Zn⁺⁺-Binding Peptides

Zn⁺⁺ ion binding with alloferon 1 (A1) and its homologs was studied by the method described [Shi Y., Beger R. D., Berg J. M. Metal binding properties of single amino acid deletion mutants of zinc finger peptides: studies using cobalt(II) as a spectroscopic prob. Biophys. J., 1993, Vol. 64, p. 749-753]. Zn⁺⁺ ion binding with A1 peptide was studied by the light-scattering method using ISS, Campaign, IL fluorimeter at 400 nm and excitation light 398 nm.

FIG. 4 shows graphs of Zn⁺⁺ ion interacting with A1 peptide.

For analysis conditions refer to Shi Y. et al. (1993)

A—(open circles) A1 and Zn(N0₃)₂. interaction Excess molar quantity of Zn⁺⁺ ion comparing to peptide was 1:10. Firm line—peptide enrichment with Zn⁺ ion. Ground peptide mass changed into aggregates with complete enrichment. EDTA was added to aggregates. Subsequent to addition of EDTA the complex quickly dissociated and peptide (alloferon) changed to soluble phase.

FIG. 4 shows that Zn⁺⁺ (Zn(NO₃)₂ reacts with A1 peptide, resulting in exponential increase of light diffusion and followed by peptide aggregation in the form of polydisperse nanoparticles up to 50-60 nm in diameter followed by formation of suspending coarse aggregates. When adding EDTA chelating agent aggregates and A1 peptide complexes are dissolved.

In this wise, A1 peptide can react with Zn⁺⁺ ion forming soluble complexes at the first stage.

Example 4. Peptides React with Zn⁺⁺ Showing High Affinity with Nickel Adsorbents

Chromatography at HiTrap columns showed that A1 acts as olygohistidine and has quite high affinity with the present adsorbent, and is completely eluted with imidazole solution. Elution was performed with gradient phosphate buffer/0.5 M imidazole (FIG. 5).

Example 5. Type I Interferons Induction

Type I interferons induction was studied by the previously published method [F. I. Yershov., O. I. Kiselev. Interferons and their inducers (from the molecule to the drug) M.: Publ. House. Geotar-Media, 2005-P. 356, Chernysh et al. 2002]. FIG. 6 shows findings for drug tests studying I type interferons induction ability. As may be inferred from the given data, Zn-A1 peptide had maximum interferon induction activity. Zn-A2 peptide was somewhat inferior. Nonmodified A1 peptide showed quite high level of interferon induction ability, but it was significantly inferior to derivatives in complex with Zn⁺⁺ ion and matched cycloferon activity.

Example 6 illustrates that these data correlate with protective action of drugs in case of nonsurvivable death grippal infection in mice.

Example 6. Antiviral Activity of the Experimental Lethal Grippal Pneumonia in White Mice, Induced with a Virus Influenza

The model of lethal grippal infection of white scrub mice of both genders with weight 10-12 g from Rappolovo nursery was used for testing of peptide complexes antiviral activity. A/Aichi/2/68 (H3N2) flu strain has been used in the work, adapted to white mice in laboratory conditions with high pathogenicity, inducing infection with developing pneumonia and lethal outcome during 5-10 days depending on the viral dose.

Peptides and their derivatives were once administered abdominally to animals 6 and 12 hours before contamination in the amount of 1-2 μg/kg of animal weight. NSS or phosphate buffer in equal volume was placebo in control animal group.

Virus was previously titrated on animals and lethal concentration for mice has been determined. The animals were exposed to virus intranasally with slight ether anesthesia in the dose of 0.2 and 5 LD₅₀. Each study group comprised 10 mice. The animals were observed during 15 days, i.e. the term when 100% animal death is observed in experimental flu. Weight and death of animals was recorded day-to-day in control and experimental groups. Based on received mortality data, mortality rates in each group (number of died for 15 days animals to total amount of contaminated animals in the group ratio), protective index. The findings are represented in the FIG. 5. Analysis of findings showed that the action of studied drugs A1 relative to influenza A virus, pathogenic for mice was comparable to efficiency of the protective effect of reference drug Remantadin (80-87%—with dose of virus 1 LD₅₀). High protective effect of Zn-A1 complexes proves that formation of Zn⁺⁺ complex with A1 significantly potentiates type A1 peptides activity. Testing method, used in this case, proves that protective effect mainly should be attributed to interferon induction. The drug showed maximum activity when using in preventive scheme.

FIG. 7 shows protective effect of the studied drugs in lethal grippal infections of mice. Based on the above, we can state that the designed peptide has all the claimed properties.

Histidine-rich peptide complexes, primarily alloferon family peptides with Zn⁺⁺ ion, will make it possible to create drugs with directed mechanism of action and design them with regard to understanding of peptide properties and composition, and drug target structure. 

What is claimed:
 1. A peptide complex organized in three-dimensional structure and characterized by general structural formula:

wherein: X₁ is selected from the group consisting of Gln, Ser, Asn, Val, Ala, Phe, and Asp or alternatively is absent; and R₁ and R₂ and comprise peptide chains that interact with transition metal ions, further wherein R₁ is selected from the group consisting of His-Gly-Val-Ser-Gly- (SEQ ID NO: 30), Cys-Val-Val-Thr-Gly- (SEQ ID NO: 31), Cys-Gly-, Val-Ser-Gly-, and His-Gly- or alternatively is absent; and R₂ is selected from the group consisting of: -Val-His-Gly, -Val-Phe-Val, -Val-His, and -Val-Asp or alternatively is absent, further wherein if X₁ is absent, R₂ must be present and if R₂ is absent, X₁ must be present.
 2. The peptide complex according to claim 1, wherein said peptide complex induces interferon synthesis.
 3. The peptide complex according to claim 1, wherein said peptide complex has antiviral activity.
 4. The peptide complex according to claim 1, wherein X₁ is present and is selected from the group consisting of Gln, Ser, Asn, Val, Ala, Phe, and Asp.
 5. The peptide complex according to claim 1, wherein R₂ is present and selected from the group consisting of: -Val-His-Gly, -Val-Phe-Val, -Val-His, and -Val-Asp.
 6. The peptide complex according to claim 1, wherein X₁, R₁, and R₂ are all present.
 7. The peptide complex according to claim 1, wherein the peptide component is 7 to 13 amino acid residues long.
 8. The peptide complex according to claim 1, wherein X₁ is Gln, R₁ is His-Gly-Val-Ser-Gly- (SEQ ID NO: 30), and R₂ is -Val-His-Gly.
 9. A peptide complex organized in three-dimensional structure and characterized by general structural formula:

wherein: X₁ is selected from the group consisting of Gln, Ser, Asn, Val, Ala, Phe, and Asp or alternatively is absent; and R₁ and R₂ comprise peptide chains that interact with transition metal ions, further wherein R₁ is selected from the group consisting of: His-Gly-Val-Ser-Gly- (SEQ ID NO: 30), Cys-Val-Val-Thr-Gly- (SEQ ID NO: 31), Cys-Gly-, and Val-Ser-Gly-, or alternatively is absent; and R₂ is selected from the group consisting of: -Val-His-Gly, -Val-Phe-Val, -Val-His, and -Val-Asp or alternatively is absent. 